Novel compounds

ABSTRACT

Novel NH-substituted fused triazoles are inhibitors of the bacterial enzyme  S aureus  methionyl t RNA synthetase and are of use in treating bacterial infections.

The present invention relates to novel NH-substituted fused triazoleswhich are inhibitors of methionyl t-RNA synthetase (MRS), processes fortheir preparation and their use in therapy as anti-bacterial agents.

t-RNA synthetases are involved in protein biosynthesis so thatinhibition thereof may be expected to lead to a cessation of cellgrowth. Thus, for instance, the compound mupirocin, produced by theorganism Pseudomonas fluorescens, is an anti-bacterial agent and is usedas the active ingredient in the product Bactroban, marketed bySmithKline Beecham. Mupirocin has been shown to be an inhibitor of theisoleucyl t-RNA synthetase. Each t-RNA synthetase represents a separatetarget for drug discovery. t-RNA synthetase inhibitors which areselective for bacterial cells over mammalian cells are of considerabletherapeutic interest as they have the potential to be used asanti-bacterial agents.

The sequence of the t-RNA synthetase genes in organisms such as S aureushave recently been determined, see for instance European Patentapplication no 97300317.1 (SmithKline Beecham, S aureus MRS), therebyassisting the process of identifying inhibitors.

WO 99/ and WO 00/21949 (SmithKline Beecham, published after the prioritydate of the present application) describe a class of 2-(NH— orO-substituted) quinolones which are potent inhibitors of methionyl t-RNAsynthetase

We have now found a further class of compounds which are potentinhibitors of methionyl t-RNA synthetase viz NH-substituted fusedtriazoles.

Accordingly, the present invention provides a compound of the formula(I):

in which:

R¹ is an optionally substituted aryl or an optionally substitutedheteroaryl ring;

X is CH₂ or CHR³ in which R³ is C₍₁₋₆₎alkyl or is linked to the orthoposition of an aryl or heteroaryl ring of R¹ to form a 5 to 7 memberedring optionally including oxygen or nitrogen as a ring atom;

Y is C₍₁₋₃₎alkylene or C₍₄₋₆₎cycloalkylene;

Z¹, Z² and Z³ is each independently selected from N or CR⁴ in which R⁴is hydrogen or a substitutent selected from halogen, cyano, (C₁-₆)alkyl,mono to perfluoro(C₁-₃)alkyl, (C₃-₇)cycloalkyl, (C₂-₆)alkenyl,(C₁-₆)alkoxy, (C₂-₆)alkenoxy, arylC₍₁₋₆₎alkoxy, halo(C₁-₆)alkyl,hydroxy, amino, mono- or di-(C₁-₆)alkylamino, acylamino, nitro, carboxy,(C₁-₆)alkoxycarbonyl, (C₁-₆)alkenyloxycarbonyl,(C₁-₆)alkoxycarbonyl(C₁-₆)alkyl, carboxy(C₁-₆)alkyl,(C₁-₆)alkylcarbonyloxy, carboxy(C₁-₆)alkyloxy,(C₁-₆)alkoxycarbonyl(C₁-₆)alkoxy, (C₁-₆)alkylthio, (C₁-₆)alkylsulphinyl,(C₁-₆)alkylsulphonyl, sulphamoyl, mono- and di-(C₁-₆)-alkylsulphamoyl,carbamoyl, mono- and di-(C₁-₆)alkylcarbamoyl, and heterocyclyl; or

a tautomer thereof, and

salts thereof, preferably pharmaceutically acceptable salts thereof.

Compounds of formula (I) are inhibitors of S aureus methionyl tRNAsynthetase.

Representative examples of R¹ when aryl include phenyl and naphthyl,each of which may be optionally substituted with up to foursubstituents. Representative examples of such substituents includeC₍₁₋₆₎ alkyl, C₍₁₋₆₎ alkoxy, C₍₁₋₆₎ alkylthio, halo, cyano, amino,sulphamoyl, phenylcarbonyl, aryl, and benzyloxy. Preferably, the phenylor naphthyl is substituted by two or three lipophilic substituents suchas chloro, bromo, iodo, methyl, methoxy, ethoxy, allyloxy, phenethyloxyor trifluoromethyl.

Representative examples of R¹ when heteroaryl include pyrrolyl, thienyl,furanyl, pyridyl, quinolinyl, benzofuranyl, and indolyl, each of whichmay be optionally substituted with up to three substituents. Preferably,the heteroaryl ring is substituted by two or three lipophilicsubstituents such as chloro, bromo, iodo, methyl, methoxy ortrifluoromethyl. Representative examples of such substituents includehalo.

Preferred examples of aryl and heteroaryl groups for R¹ include phenyl,indolyl and thienyl.

Representative examples of X include CH₂ or forming with R² a5-7-membered ring fused to an aryl or heteroaryl ring, preferablyincluding oxygen or nitrogen as a ring atom, for instance chroman-4-yland 1,2,3,4-tetrahydroquinolin-4-yl in which R² is phenyl

Representative examples of R¹X include benzyl, chroman-4-yl,1,2,3,4-tetrahydroquinolin-4-yl, indol-7-ylmethyl, and thien-2-ylmethylin which the aryl/heteroaryl ring may be optionally substituted ashereinbefore defined. Preferably, R¹X is optionally substituted benzyl,indol-7-ylmethyl or thien-2-ylmethyl.

Representative examples of Y include a C₂ alkylene chain or a1,2-cyclopentylene group. Preferably, Y is a C₂ alkylene chain.

Preferably, only one of Z¹, Z² and Z³ is N and the other two are CR⁴.Preferably, Z¹ is N and Z² and Z³ is each CH. Preferably, the fusedheteroaryl ring comprising a triazole ring is1H-pyrazolo[1,5-b][1,2,4]triazole.

Salts may be formed from inorganic and organic acids. Representativeexamples of suitable inorganic and organic acids from whichpharmaceutically acceptable salts of compounds of formula (I) may beformed include maleic, fumaric, benzoic, ascorbic, pamoic, succinic,bismethylenesalicylic, methanesulfonic, ethanedisulfonic, acetic,propionic, tartaric, salicylic, citric, gluconic, aspartic, stearic,palmitic, itaconic, glycolic, p-aminobenzoic, glutamic, benzenesulfonic,hydrochloric, hydrobromic, sulfuric, cyclohexylsulfamic, phosphoric andnitric acids.

When used herein, the term “alkyl” and similar terms such as “alkoxy”includes all straight chain and branched isomers. Representativeexamples thereof include methyl, ethyl, n-propyl, iso-propyl, n-butyl,sec-butyl, iso-butyl, t-butyl, n-pentyl and n-hexyl.

Preferred substituents for an alkyl group include, for example, andunless otherwise defined, halogen, cyano, azido, nitro, carboxy,(C₁₋₆)alkoxycarbonyl, carbamoyl, mono- or di-(C₁₋₆)alkylcarbamoyl,sulpho, sulphamoyl, mono- or di-(C₁₋₆)alkylsulphamoyl, amino, mono- ordi-(C₁₋₆)alkylamino, acylamino, ureido, (C₁₋₆)alkoxycarbonylamino,2,2,2-trichloroethoxycarbonylamino, aryl, heterocyclyl, hydroxy,(C₁-6)alkoxy, acyloxy, oxo, acyl, 2-thienoyl, (C₁₋₆)alkylthio,(C₁₋₆)alkylsulphinyl, (C₁₋₆)alkylsulphonyl, hydroxyimino,(C₁₋₆)alkoxyimino, hydrazino, hydrazono, benzohydroximoyl, guanidino,amidino and iminoalkylamino.

When used herein, the term “aryl” includes, unless otherwise defined,phenyl or naphthyl optionally substituted with up to five, preferably upto three substituents.

When substituted, an aryl group may have up to three substituents.Preferred substituents for an aryl group include, for example, andunless otherwise defined, halogen, cyano, (C₁-₆)alkyl, mono toperfluoro(C₁-₃)alkyl, (C₃-₇)cycloalkyl, (C₂-₆)alkenyl, (C₁-₆)alkoxy,(C₂-₆)alkenoxy, arylC₍₁₋₆₎alkoxy, halo(C₁-₆)alkyl, hydroxy, amino, mono-or di-(C₁-₆)alkylamino, acylamino, nitro, carboxy, (C₁-₆)alkoxycarbonyl,(C₁-₆)alkenyloxycarbonyl, (C₁-₆)alkoxycarbonyl(C₁-₆)alkyl,carboxy(C₁-₆)alkyl, (C₁-₆)alkylcarbonyloxy, carboxy(C₁-₆)alkyloxy,(C₁-₆)alkoxycarbonyl(C₁-₆)alkoxy, (C₁-₆)alkylthio, (C₁-₆)alkylsulphinyl,(C₁-₆)alkylsulphonyl, sulphamoyl, mono- and di-(C₁-₆)-alkylsulphamoyl,carbamoyl, mono- and di-(C₁-₆)alkylcarbamoyl, and heterocyclyl.

When used herein, the term “heteroaryl” includes single or fused ringscomprising up to four hetero-atoms in the ring selected from oxygen,nitrogen and sulphur and optionally substituted with up to threesubstituents. Preferably the heteroaryl ring comprises from 4 to 7,preferably 5 to 6, ring atoms. A fused heteroaryl ring system mayinclude carbocyclic rings and need only include one heterocyclic ring.

When used herein, the term “heterocyclyl” includes aromatic andnon-aromatic single or fused rings comprising up to four hetero-atoms inthe ring selected from oxygen, nitrogen and sulphur and optionallysubstituted with up to three substituents. Suitably the heterocyclicring comprises from 4 to 7, preferably 5 to 6, ring atoms. A fusedheterocyclic ring system may include carbocyclic rings and need onlyinclude one heterocyclic ring.

When substituted, a heteroaryl or a heterocyclyl group may have up tothree substituents. Preferred such substituents include those previouslymentioned for an aryl group as well as oxo.

When used herein, the terms ‘halogen’ and ‘halo’ include fluorine,chlorine, bromine and iodine and fluoro, chloro, bromo and iodo,respectively.

The compounds according to the invention are suitably provided insubstantially pure form, for example at least 50% pure, suitably atleast 60% pure, advantageously at least 75% pure, preferably at least85% pure, more preferably at least 95% pure, especially at least 98%pure, all percentages being calculated as weight/weight. An impure orless pure form of a compound according to the invention may, forexample, be used in the preparation of a more pure form of the samecompound or of a related compound (for example a correspondingderivative) suitable for pharmaceutical use.

Preferred compounds of formula (I) include:

-   2-[3-(4,5-Dibromo-3-methylthien-2-ylmethylamino)propylamino]-1H-pyrazolo[1,5-b][1,2,4]triazole.

A compound of formula (I) may be prepared by reacting a compound offormula (II):

in which Y, Z¹, Z² and Z³ are as hereinbefore defined;with either:

(a) for a compound of formula (I) in which X is CH₂, an aldehyde offormula (III):R¹CHO   (III)in which R¹ is as hereinbefore defined;under reductive alkylation conditions;

(b) for a compound of formula (I) in which X is CH₂ substituted byC₍₁₋₆₎ alkyl or in which R¹ and X are linked by a 5-7-membered ringoptionally containing oxygen or nitrogen, a ketone of formula (IV):R¹R³CO   (IV)in which R¹ and R³ are as hereinbefore defined;under reductive alkylation conditions.

Suitable reductive alkylating conditions are well known in the art andinclude for instance, the use of sodium triacetoxyborohydride in asolvent system such as DMF/acetic acid or sodium cyanoborohydride inmethanol/acetic acid. Reductive alkylation with an aldehyde is typicallycarried out at room temperature for a period of 1-16 h. Reductivealkylation with a ketone is typically carried out in refluxing methanolfor a period of 16-40 h.

A compound of formula (II) may be prepared from a corresponding compoundof formula (V):

in which Z¹, Z² and Z³ are as hereinbefore defined;by cyclisation with a protected isocyanate of the formula (VI):BocNHYCH₂NCS   (VI)in which Boc is benzyloxycarbonyl,followed by removal of the Boc protecting group.

Triazole compounds of formula (V) are either already known in the art orcan be prepared by analogy with standard known methods for preparingsuch ring systems.

The compounds of this invention are active against both Gram negativeand Gram positive organisms, including Haemophilus, for instance H.influenzae Q1; Moraxella, for instance M catarrhalis 1502; Streptococci,for instance S. pyogenes CN10 and S. pneumoniae R6; Staphylococci, forinstance S. aureus Oxford; Escherichia, for instance E. Coli DC0, andEnterococci, for instance Ent. faecelis I. In addition, compounds ofthis invention are active against Staphylococci organisms such as S.aureus and coagulase-negative strains of Staphylocci such as S.epidermidis which are resistant (including multiply-resistant) to otheranti-bacterial agents, for instance, β-lactam antibiotics such as, forexample, methicillin; macrolides; aminoglycosides, and lincosamides.Compounds of the present invention are therefore useful in the treatmentof MRSA, MRCNS and MRSE. Compounds of the present invention are alsoactive against strains of E. faecalis including vancomycin resistantstrains and therefore of use in treating infections associated with VREorganisms. Furthermore, compounds of the present invention are useful inthe treatment of Staphylococci organisms which are resistant tomupirocin.

Bacterial infections which may be treated include respiratory tractinfections, otitis media, meningitis, endocarditis, skin and soft tissueinfections in man, mastitis in cattle, and respiratory infections inanimals such as pigs and cattle. Accordingly, in a further aspect, thepresent invention provides a method of treating bacterial infection inhuman or non-human animals, which method comprises administering atherapeutically effective amount of a compound of formula (I) ashereinbefore defined, to a human or non-human animal in need of suchtherapy.

The present invention provides a pharmaceutical composition comprising acompound of formula (I) together with a pharmaceutically acceptablecarrier or excipient.

The present invention also provides a method of treating bacterialinfections in animals, especially in humans and in domesticated mammals,which comprises administering a compound of formula (I), or acomposition according to the invention, to a patient in need thereof.

The invention further provides the use of a compound of formula (I) inthe preparation of a medicament composition for use in the treatment ofbacterial infections.

The compounds and compositions according to the invention may beformulated for administration in any convenient way for use in human orveterinary medicine, by analogy with other antibiotics.

The compounds and compositions according to the invention may beformulated for administration by any route, for example oral, topical orparenteral. The compositions may, for example, be made up in the form oftablets, capsules, powders, granules, lozenges, creams, syrups, orliquid preparations, for example solutions or suspensions, which may beformulated for oral use or in sterile form for parenteral administrationby injection or infusion.

Tablets and capsules for oral administration may be in unit dosage form,and may contain conventional excipients including, for example, bindingagents, for example, syrup, acacia, gelatin, sorbitol, tragacanth, orpolyvinylpyrrollidone; fillers, for example lactose, sugar,maize-starch, calcium phosphate, sorbitol or glycine; tablettinglubricants, for example magnesium stearate, talc, polyethylene glycol orsilica; disintegrants, for example potato starch; and pharmaceuticallyacceptable wetting agents, for example sodium lauryl sulphate. Thetablets may be coated according to methods well known in normalpharmaceutical practice.

Oral liquid preparations may be in the form of, for example, aqueous oroily suspensions, solutions, emulsions, syrups or elixirs, or may bepresented as a dry product for reconstitution with water or anothersuitable vehicle before use. Such liquid preparations may containconventional additives, including, for example, suspending agents, forexample sorbitol, methyl cellulose, glucose syrup, gelatin, hydroxyethylcellulose, carboxymethyl cellulose, aluminium stearate gel orhydrogenated edible fats; emulsifying agents, for example lecithin,sorbitan monooleate or acacia; non-aqueous vehicles (which may includeedible oils), for example almond oil, oily esters (for exampleglycerine), propylene glycol, or ethyl alcohol; preservatives, forexample methyl or propyl p-hydroxybenzoate or sorbic acid; and, ifdesired, conventional flavouring and colour agents.

Compositions according to the invention intended for topicaladministration may, for example, be in the form of ointments, creams,lotions, eye ointments, eye drops, ear drops, impregnated dressings, andaerosols, and may contain appropriate conventional additives, including,for example, preservatives, solvents to assist drug penetration, andemollients in ointments and creams. Such topical formulations may alsocontain compatible conventional carriers, for example cream or ointmentbases, and ethanol or oleyl alcohol for lotions. Such carriers mayconstitute from about 1% to about 98% by weight of the formulation; moreusually they will constitute up to about 80% by weight of theformulation.

Compositions according to the invention may be formulated assuppositories, which may contain conventional suppository bases, forexample cocoa-butter or other glycerides.

Compositions according to the invention intended for parenteraladministration may conveniently be in fluid unit dosage forms, which maybe prepared utilizing the compound and a sterile vehicle, water beingpreferred. The compound, depending on the vehicle and concentrationused, may be either suspended or dissolved in the vehicle. In preparingsolutions, the compound may be dissolved in water for injection andfilter-sterilised before being filled into a suitable vial or ampoule,which is then sealed. Advantageously, conventional additives including,for example, local anaesthetics, preservatives, and buffering agents canbe dissolved in the vehicle. In order to enhance the stability of thesolution, the composition may be frozen after being filled into thevial, and the water removed under vacuum; the resulting dry lyophilizedpowder may then be sealed in the vial and a accompanying vial of waterfor injection may be supplied to reconstitute the liquid prior to use.Parenteral suspensions may be prepared in substantially the same mannerexcept that the compound is suspended in the vehicle instead of beingdissolved and sterilisation cannot be accomplished by filtration. Thecompound may instead be sterilised by exposure to ethylene oxide beforebeing suspended in the sterile vehicle. Advantageously, a surfactant orwetting agent is included in such suspensions in order to facilitateuniform distribution of the compound.

A compound or composition according to the invention may suitably beadministered to the patient in an antibacterially effective amount.

A composition according to the invention may suitably contain from 0.1%by weight, preferably from 10 to 60% by weight, of a compound accordingto the invention (based on the total weight of the composition),depending on the method of administration.

The compounds according to the invention may suitably be administered tothe patient at a daily dosage of from 1.0 to 50 mg/kg of body weight.For an adult human (of approximately 70 kg body weight), from 50 to 3000mg, for example about 1500 mg, of a compound according to the inventionmay be administered daily. Suitably, the dosage for adult humans is from5 to 20 mg/kg per day. Higher or lower dosages may, however, be used inaccordance with normal clinical practice.

When the compositions according to the invention are presented in unitdosage form, each unit dose may suitably comprise from 25 to 1000 mg,preferable from 50 to 500 mg, of a compound according to the invention.

The following Examples illustrate the present invention.

General method for reductive amination To a suspension of the amine (0.2mmol) (containing 0.5 mmol sodium acetate if the amine was present asthe dihydrochloride) in methanol (2 ml) was added the aldehyde (0.2mmol) in methanol (2 ml) and acetic acid (0.033 ml). After stirringunder argon for 10 min, NaCNBH₃ (24 mg, 0.4 mmol) in MeOH (1 ml) wasadded and the reaction stirred for 16 h. The reaction mixture wasapplied to a 2 g Varian Bond Elute SCX cartridge which was flushed withMeOH (8 ml). The cartridge was then eluted with 8 ml 0.2 M NH₃ in MeOH,and this eluate evaporated to dryness. The residue was purified bychromatography on silica gel eluting with 2-10% (9:1 MeOH/20 M NH₃) inCH₂Cl₂. Product-containing fractions were combined and evaporated underreduced pressure to give the product as a white solid. To convert thisinto the corresponding dihydrochloride, the solid was dissolved in 1.0 MHCl in methanol (0.4 ml) and the solution evaporated to dryness.

An alternative method using polymer-supported cyanoborohydride was alsoused. (Polystyrylmethyl)trimethylammonium cyanoborohydride (Novobiochem)(3.64 mmol/g, 100 mg) was used in place of sodium cynoborohydride. Thereaction was worked up by filtration, evaporation. followed bychromatography on silica gel as described above.

Intermediate1—N¹-(1H-Pyrazolo[1,5-b][1,2,4]triazol-2-yl)propane-1,3-diamine

a) [3-(1H-Pyrazolo[1,5-b][1,2,4]triazol-2-ylamino)propyl]carbamic acidtert-butyl ester: A mixture of 1,5-diaminopyrazole (72 mg; Synthesis1986, 71-74), (3-isothiocyanatopropyl)carbamic acid tert-butyl ester(158 mg), 4-(dimethylamino)pyridine (18 mg), and dry 1,2-dichloroethanewas sonicated until a homogenous solution was obtained and then kept at70° C. for 18 h. The solvent was removed in vacuo and the residuesubmitted to column chromatography to yield a thiourea (80 mg yellowsolid: m/z (ESI) 337 (MNa⁺, 40%), 313 ([M−H]⁻, 51%). This thiourea (75mg) was dissolved in dichloromethane (2.5 ml) and acetonitrile (2 ml)containing N,N-diisopropylethylamine (0.13 ml) and4-(dimethylamino)pyridine (5 mg). To methanesulfonyl chloride (0.37 ml)was added dichloromethane to a volume of 10 ml, and of this solution wasadded dropwise at 25° C. to the reaction mixture 1.0 ml, plus 0.1 mlafter 15 min, plus 0.1 ml after 1 h. After an additional 15 min theresulting mixture was concentrated and the title compound isolated bycolumn chromatography: yellow oil (39 mg), m/z (ESI) 279 ([M−H]⁻, 21%).

b) N¹-(1H-Pyrazolo[1,5-b][1,2,4]triazol-2-yl)propane-1,3-diamine:[3-(1H-Pyrazolo[1,5-b][1,2,4]triazol-2-ylamino)propyl]carbamic acidtert-butyl ester was allowed to react with trifluoroacetic acid (0.5 ml)for 30 min at 25° C. Volatiles were evaporated and the residue submittedto column chromatography to give the title compound (22 mg) as a yellowfilm: m/z (ESI) 179 ([M−H]⁻, 100%).

EXAMPLE 12-[3-(3-Chloro-5-methoxy-1H-indol-7-ylmethylamino)propylamino]-1H-pyrazolo[1,5-b][1,2,4]triazoledihydrochloride

According to the general procedure for reductive amination3-chloro-5-methoxy-1H-indol-7-carbaldehyde (26 mg) was allowed to reactwith Intermediate 1 (20 mg). The product was isolated by columnchromatography, treated with excess HCl in methanol, the volatilesevaporated, and the residue triturated with dichloromethane to give thetitle compound (7 mg) as an orange solid: m/z (ESI from a solution ind₄-MeOH) 372 ([M−H]⁻, 41%), 373 ([M+D−2H]⁻, 100%).

EXAMPLE 22-[3-(4,5-Dibromo-3-methylthien-2-ylmethylamino)propylamino]-1H-pyrazolo[1,5-b][1,2,4]triazole dihydrochloride

According to the general procedure for reductive amination4,5-dibromo-3-methylthiophen-2-carbaldehyde (85 mg) was allowed to reactwith Intermediate 1 (88 mg). Treatment with excess HCl in methanolprovided the title compound (15 mg) as a white solid: m/z (ESI MeOH) 448([MH]⁺, 100%).

Biological Data

1. Enzyme Inhibition (S. aureus MRS)—Aminoacylation Assay

Compounds of the present invention may be assayed for their ability toinhibit the enzyme methionyl tRNA synthetase (MRS), using recombinant S.aureus MRS, as follows:

Reaction Mix (per 1 ml) Stock Volume (ul) Final Concentration 100 mMTris/Cl, pH 7.9 600 30 mM 250 mM KCl 75 mM 125 mM ATP 40 2.5 mM 250 mMMgCl₂ 80 10 mM 50 mM DTT 80 2 mM 0.5 mM Met (S-35 hot and cold) 40 10 uMSolid tRNA 4 mg/ml 2 mg/ml (Mixed E. coli MRE 600) H₂O 160 10 ×Inhibitor (0-100 μM) 5 μl per well 0-10 μM

The reaction is started by adding 20 μl appropriately diluted pureenzyme (pre-incubated with inhibitor) to 25 μl reaction mix for 10 minat room temperature. The reaction is terminated by the addition of 100μl 5% trichloroacetic acid, 10% glycerol. The TCA precipitate isharvested onto dry Unifilter GFC plates using a Packard Filtermate CellHarvester. The filters are washed with 4×200 μl of 50% industrialmethylated spirit, before drying. 30 μl of Microscint 20 is added toeach well and plates are counted on a TopCount. (Packard 96 wellcounter).

Reagents

Mixed E. coli MRE 600 tRNA and ATP were purchased fromBoehringer-Mannheim, L-[³⁵ S] methionine from Amersham and otherreagents from Sigma.

Pure recombinant S. aureus MRS (EP application number 97300317.1,SmithKline Beecham) was obtained using standard purification procedures.The enzyme is diluted in Dilution Buffer which consists of 10 mMTris/Cl, 2 mM DTT pH 7.9.

Results

Examples 1 and 2 have IC₅₀ values against S. aureus MRS of <10 nM. Allare highly selective with respect to the mammalian enzyme (no inhibitionof rat MRS up to 1 μM).

2. Enzyme Inhibition (H. influenzae MRS)—Aminoacylation Assay

Compounds of the present invention may be assayed for their ability toinhibit the enzyme methionyl tRNA synthetase (MRS), using recombinant H.influenzae MRS (R. D. Fleischmann et al., Science, 269, 496-512, 1995),as follows:

Reaction Mix (per 1 ml) Stock Volume (μl) Final Concentration 100 mMTris/Cl, pH 7.9 600 30 mM 250 mM KCl 75 mM 125 mM ATP 40 2.5 mM 250 mMMgCl₂ 80 10 mM 50 mM DTT 80 2 mM 1 mM Met (H-3 hot and cold) 20 10 μMSolid tRNA 4 mg/ml 2 mg/ml (Mixed E. coli MRE 600) H₂O 180 10 ×Inhibitor (0-100 μM) 5 μl per well 0-10 μM

The reaction is started by adding 20 μl appropriately diluted pureenzyme (pre-incubated with inhibitor) to 25 μl reaction mix for 10 minat room temperature. The reaction is terminated by the addition of 150μl 167 mM sodium citrate, pH 2.15 containing phosphodiesterase (PDE) SPAbeads (0.833 mg/ml). The binding of the radiolabelled product to thebead brings the isotope into close enough proximity to allow radiationfrom the tritium to excite the scintillant within the bead. Any unboundradiolabel is not close enough to the scintillant to allow this energytransfer, so no signal is generated. Following termination of thereaction, plates are spun at 2500 rpm for 5 min in a Mistral 300E platecentrifuge (or alternatively allowed to stand for 1 hour). The assay isconducted in 96-well Optiplates (Packard). Plates are counted on aTopCount. (Packard 96 well counter).

Reagents

Mixed E. coli MRE 600 tRNA and ATP were purchased fromBoehringer-Mannheim, L-[methyl-³H]methionine and phosphodiesterasescintillation proximity (SPA) beads from Amersham Pharmacia Biotech andother reagents from Sigma.

3. Antibacterial Activity

Compounds of the present invention were assayed for antibacterialactivity against a range of pathogenic organisms (strains of S aureus,Spneumoniae, Efaecalis, H influenzae and M catarrhalis) in a standardMIC assay modified by the inclusion of cyclodextrin, to assist withsolubility.

Example 1 had MIC's<32 μg/ml against some strains of the organisms S.aureus, S. pneumoniae, and M Catarrhalis, and E. faecalis.

Example 2 was also active against H. influenzae.

1. A compound of formula (I):

in which: R¹ is an optionally substituted aryl or an optionallysubstituted heteroaryl ring; X is CH₂ or CHR³ in which R³ is C₍₁₋₆₎alkylor is linked to the ortho position of an aryl or heteroaryl ring of R¹to form a 5 to 7 membered ring optionally including oxygen or nitrogenas a ring atom; Y is C₍₁₋₃₎alkylene or C₍₄₋₆₎cycloalkylene; Z¹, Z² andZ³ is each independently selected from N or CR⁴ in which R⁴ is hydrogenor a substitutent selected from halogen, cyano, (C₁-₆)alkyl, mono toperfluoro(C₁-₃)alkyl, (C₃-₇)cycloalkyl, (C₂-₆)alkenyl, (C₁-₆)alkoxy,(C₂-₆)alkenoxy, arylC₍₁₋₆₎alkoxy, halo(C₁-₆)alkyl, hydroxy, amino, mono-or di-(C₁-₆)alkylamino, acylamino, nitro, carboxy, (C₁-₆)alkoxycarbonyl,(C₁-₆)alkenyloxycarbonyl, (C₁-₆)alkoxycarbonyl(C₁-₆)alkyl,carboxy(C₁-₆)alkyl, (C₁-₆)alkylcarbonyloxy, carboxy(C₁-₆)alkyloxy,(C₁-₆)alkoxycarbonyl(C₁-₆)alkoxy, (C₁-₆)alkylthio, (C₁-₆)alkylsulphinyl,(C₁-₆)alkylsulphonyl, sulphamoyl, mono- and di-(C₁-₆)-alkylsulphamoyl,carbamoyl, mono- and di-(C₁-₆)alkylcarbamoyl, and heterocyclyl; or atautomer thereof, and salts thereof, preferably pharmaceuticallyacceptable salts thereof.
 2. A compound of formula (I) as claimed inclaim 1 in which R¹ when aryl is selected from phenyl and naphthyl, eachof which may be optionally substituted with up to four substituents; orwhen heteroaryl is selected from pyrrolyl, thienyl, furanyl, pyridyl,quinolinyl, benzofuranyl, and indolyl, each of which may be optionallysubstituted with up to three substituents.
 3. A compound of formula (I)as claimed in claim 1 or 2 in which aryl and heteroaryl groups for R¹are phenyl and indolyl and thienyl, respectively.
 4. A compound offormula (I) as claimed any one of claims 1 to 3 in which X is CH₂ orforms with R² a 5-7-membered ring fused to an aryl ring or a heteroarylring which includes oxygen or nitrogen as a ring atom.
 5. A compound offormula (I) as claimed any one of claims 1 to 4 in which R¹X is benzyl,chroman-4-yl, 1,2,3,4-tetrahydroquinolin-4-yl, indol-7-ylmethyl, andthien-2-ylmethyl in which the aryl/heteroaryl ring may be optionallysubstituted.
 6. A compound of formula (I) as claimed any one of claims 1to 5 in which Y is a C₂ alkylene chain.
 7. A compound of formula (I) asclaimed in any one of claims 1 to 6 in which only one of Z¹, Z² and Z³is N and the other two are CR⁴.
 8. A compound of formula (I) as claimedin claim 7 in which Z¹ is NH and Z² and Z³ is each CH.
 9. Apharmaceutical composition comprising an antibacterially effectiveamount of a substance or compound of formula (I) as claimed in claim 1together with a pharmaceutically acceptable carrier or excipient.
 10. Acompound of formula (I) as claimed in for use in therapy.
 11. A compoundof formula (I) as claimed in for use in the treatment of bacterialinfections.
 12. Use of a compound of formula (I) as claimed in claim 1in the manufacture of a medicament for use in the treatment of bacterialinfections.
 13. A process for preparing a compound of formula (I) asclaimed in claim 1 which process comprises (i) reacting a compound offormula (II):

in which Y, Z¹, Z² and Z³ are as hereinbefore defined; with either: (a)for a compound of formula (I) in which X is CH₂, an aldehyde of formula(III):R¹CHO   (III) in which R¹ is as hereinbefore defined; under reductivealkylation conditions; (b) for a compound of formula (I) in which X isCH₂ substituted by C₍₁₋₆₎ alkyl or in which R¹ and X are linked by a5-7-membered ring opt.cont. oxygen or nitrogen, a ketone of formula(IV):R¹R³CO   (IV) in which R¹ and R³ are as hereinbefore defined; underreductive alkylation conditions.